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1.
We sequenced and characterized the complete mitochondrial genome of the Japanese fish tapeworm D. nihonkaiense. The genome is a circular-DNA molecule of 13607 bp (one nucleotide shorter than that of D. latum mtDNA) containing 12 protein-coding genes (lacking atp8), 22 tRNA genes and two rRNA genes. Gene order and genome content are identical to those of the other cestodes reported thus far, including its congener D. latum. The only exception is Hymenolepis diminuta in which the positions of trnS2 and trnL1 are switched. We tested a PCR-based molecular assay designed to rapidly and accurately differentiate between D. nihonkaiense and D. latum using species-specific primers based on a comparison of their mtDNA sequences. We found the PCR-based system to be very reliable and specific, and suggest that PCR-based identification methods using mtDNA sequences could contribute to the study of the epidemiology and larval ecology of Diphyllobothrium species. 相似文献
2.
David Scott Clarence Clark Andrea Fyffe Michon Walker Paul Tooley John Maas Ken Deahl 《Molecular biotechnology》1998,9(3):273-276
An oligonucleotide (primer), designed from a conserved region of the multi-allelicb locus of the basidiomycete fungusUstilago maydis, generated reproducible PCR fingerprints inPhytophthora species. The primer hybridized in a species-specific manner to nucleotide sequences interspersed in the genome of the closely related members ofPhytophthora taxonomic group IV. We recommend the use of this PCR procedure as an alternative method for resolving the close taxonomic affinity of some members of the genusPhytophthora. 相似文献
3.
Sequence-tagged-site-facilitated PCR for barley genome mapping 总被引:6,自引:0,他引:6
S. Tragoonrung V. Kanazin P. M. Hayes T. K. Blake 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1992,84(7-8):1002-1008
Summary Speed, efficiency, and safety considerations have led many genome mapping projects to evaluate polymerase chain reaction (PCR) sequence amplification as an alternative to Southern blot analysis. However, the availability of informative primer sequences can be a limiting factor in PCR-based mapping. An alternative to random amplified polymorphism detection (RAPD) is the sequence-tagged-site (STS) approach. If informative primer sequences could be derived from known sequences, then current maps, which are based on both known function and anonymous clones, might be easily converted to maps utilizing PCR technology. In this paper, four pairs of primer sequences were obtained from published sequences, and four pairs were obtained by sequencing portions of DNA clones from genomic clones derived from a random genomic library used in the North American Barley Genome Mapping Project (NABGMP). These primers were used to screen for polymorphisms in the progeny of a winter x spring and a spring x spring barley cross. Two types of polymorphisms were distinguished using these primer sets: (1) insertion/deletion events that could be read directly from agarose gels, and (2) point mutation events. The latter were identified using polyacrylamide-gel electrophoresis of PCR products following digestion with restriction endonucleases (four-base cutters). To determine whether the PCR-based polymorphisms were allelic to polymorphisms identified by the clones from which the primer sequences derived, chromosomal assignments and (when possible) co-segregation analysis was performed. 相似文献
4.
Allele-specific amplification of polymorphic sites for the detection of powdery mildew resistance loci in cereals 总被引:4,自引:0,他引:4
V. Mohler A. Jahoor 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1996,93(7):1078-1082
Primers for the polymerase chain reaction (PCR) were tailored to selectively amplify RFLP marker alleles associated with resistance and susceptibility for powdery mildew in cereals. The differentiation between marker alleles for susceptible and resistant genotypes is based on the discrimination of a single nucleotide by using allele-specific oligonucleotides as PCR primers. The PCR assays developed are diagnostic for RFLP alleles at the loci MWG097 in the barley genome and Whs350 in the wheat genome. The first marker locus is closely linked to MlLa resistance in barley, while the latter is linked to Pm2 resistance locus in wheat. PCR analysis of 31 barley and 30 wheat cultivars, with some exceptions, verified the presence or absence of the resistance loci investigated. These rapid PCR-based approaches are proposed as an efficient alternative to conventional procedures for selecting powdery mildew-resistant genotypes in breeding programs. 相似文献
5.
Accurate identification of sex in birds is important for the management and conservation of avian wildlife in several ways, namely in the development of population, behavioral and ecological studies, as well as in the improvement of ex situ captive breeding programs. In general, nestlings, juveniles and adult birds of a wide number of sexually monomorphic species cannot be sexed based on phenotypic traits. The development of molecular methodologies for avian sexing overcame these difficulties, allowing a reliable gender differentiation for these species. The polymerase chain reaction (PCR)-based methods have been widely applied in molecular sexing of birds, using a large diversity of sex-linked markers. During the last 15 yrs, there was a continuous improvement in the PCR-based protocols for bird sexing, increasing the accuracy, speed and high-throughput applicability of these techniques. The recent advances in real-time PCR platforms and whole genome analysis methods provided new resources for the detection and analysis of novel specific markers and protocols. This review presents a comparative guide of classical and recent advances in PCR-based methods for avian molecular sexing, highlighting its strengths and limitations. Future research opportunities in this field are also addressed. 相似文献
6.
Physical mapping of an arabidopsis DNA sequence of interest can easily be performed by PCR. This is done by using specific primers and pooled DNA templates isolated from publicly available YAC or BAC libraries. We present simple protocols for preparing pooled YAC DNAs and PCR-based detection of sequences in them by which several sequences can be mapped in a short time. 相似文献
7.
Transgene-specific and event-specific molecular markers for characterization of transgenic papaya lines resistant to Papaya ringspot virus 总被引:1,自引:0,他引:1
Ming-Jen Fan Shu Chen Yi-Jung Kung Ying-Huey Cheng Huey-Jiunn Bau Tien-Tsai Su Shyi-Dong Yeh 《Transgenic research》2009,18(6):971-986
The commercially valuable transgenic papaya lines carrying the coat protein (CP) gene of Papaya ringspot virus (PRSV) and conferring virus resistance have been developed in Hawaii and Taiwan in the past decade. Prompt and sensitive
protocols for transgene-specific and event-specific detections are essential for traceability of these lines to fulfill regulatory
requirement in EU and some Asian countries. Here, based on polymerase chain reaction (PCR) approaches, we demonstrated different
detection protocols for characterization of PRSV CP-transgenic papaya lines. Transgene-specific products were amplified using
different specific primer pairs targeting the sequences of the promoter, the terminator, the selection marker, and the transgene,
and the region across the promoter and transgene. Moreover, after cloning and sequencing the DNA fragments amplified by adaptor
ligation-PCR, the junctions between plant genomic DNA and the T-DNA insert were elucidated. The event-specific method targeting
the flanking sequences and the transgene was developed for identification of a specific transgenic line. The PCR patterns
using primers designed from the left or the right flanking DNA sequence of the transgene insert in three selected transgenic
papaya lines were specific and reproducible. Our results also verified that PRSV CP transgene is integrated into transgenic
papaya genome in different loci. The copy number of inserted T-DNA was further confirmed by real-time PCR. The event-specific
molecular markers developed in this investigation are crucial for regulatory requirement in some countries and intellectual
protection. Also, these markers are helpful for prompt screening of a homozygote-transgenic progeny in the breeding program. 相似文献
8.
J A Cowley C M Dimmock C Wongteerasupaya V Boonsaeng S Panyim P J Walker 《Diseases of aquatic organisms》1999,36(2):153-157
Corresponding genomic regions of isolates of yellow head virus (YHV) from Thailand and gill-associated virus (GAV) from Australia were compared by RT-PCR and sequence analysis. PCR primers designed from sequences in the GAV ORF1b polyprotein gene amplified the corresponding 577 nucleotide region of the YHV genome. Comparison of the amplified region indicated 85.1% nucleotide and 95.8% amino acid sequence identity. YHV PCR primers designed to amplify a 135 nucleotide product previously described as a YHV diagnostic probe failed to amplify the corresponding product from GAV RNA. However, the cognate GAV sequence for this and another recently reported YHV sequence were located in an upstream region of the ORF1b gene. A comparison of these sequences indicated identities of 83.0 and 80.9% at the nucleotide level and 86.7 and 86.5% at the amino acid level, respectively. The data indicate that GAV and YHV are closely related but distinct viruses for which differential diagnostic probes can be applied. 相似文献
9.
Tuberculosis is an under-recognized yet catastrophic health problem, particularly in developing countries. The HIV pandemic has served to increase the number of susceptible individuals, and multidrug-resistance and poor socioeconomic conditions also augment the prevalence and the consequences of the disease. To control the disease and its spread, it is vital that tuberculosis diagnostics are accurate and rapid. Whereas microscopy and culture have several limitations (low sensitivity is a problem for the former, while the latter has a delayed turnaround time), PCR-based techniques targeting regions of the Mycobacterium tuberculosis genome such as IS6110 have proved to be useful. The purpose of this review is to assess the use of PCR-RFLP, nested PCR and real-time PCR protocols and the choice of target regions for the detection of M. tuberculosis. Real-time PCR for the detection of M. tuberculosis target genes in clinical specimens has contributed to improving diagnosis and epidemiologic surveillance in the past decade. However, targeting one genome sequence such as IS6110 may not by itself be sufficiently sensitive to reach 100% diagnosis, especially in the case of pulmonary tuberculosis. Additional testing for target genome sequences such as hsp65 seems encouraging. An interesting approach would be a multiplex real-time PCR targeting both IS6110 and hsp65 to achieve comprehensive and specific molecular diagnosis. This technology needs development and adequate field testing before it becomes the acceptable gold standard for diagnosis. 相似文献
10.
Theodoros N. Rampias 《Analytical biochemistry》2009,388(2):342-344
Here we report the development of an accurate and efficient genome walking approach based on ligation-mediated polymerase chain reaction (PCR) and magnetic capture hybrid selection technique. This approach overcomes the nonspecific amplification products that often occur in similar PCR-based methods. Our strategy was successfully applied for the cloning of the promoter region of the Cc RNase gene. This rapid, cost-effective, and sensitive protocol can easily be extended for use in the isolation and amplification of any target sequences for which only partial information is known such as identification of the position of transposable elements and integrated viral DNA sequences. 相似文献
11.
Use of 3'' untranslated sequences of human cDNAs for rapid chromosome assignment and conversion to STSs: implications for an expression map of the genome. 总被引:9,自引:1,他引:8
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A general mapping strategy is described in which the 3'untranslated regions of human cDNAs are used to design PCR primers which will selectively amplify human genomic sequences in a rodent background. When applied to panels of human x hamster somatic cell hybrid DNAs, this approach provides a PCR-based method for rapidly assigning genes to specific chromosomes and chromosomal regions. In addition, it follows from the virtual absence of introns in the 3'untranslated region of vertebrate genes that within this region the cDNA sequences almost always will be identical to those of the genomic DNA and can therefore be used to automatically generate gene-specific sequence-tagged sites (STSs). We have applied this strategy to six human cDNAs and demonstrate that 1) the primers selectively amplify human genomic DNA and 2) the PCR product is of the size predicted from the cDNA. To test this approach further we have utilized it to confirm the known chromosomal location of the retinoblastoma gene. Lastly, we describe how this strategy can readily be applied to unknown human cDNAs, and thereby be integrated into efforts to generate a human STS expression map of the genome. A strategy for production of such a map, using human brain cDNAs as a model, is described. 相似文献
12.
Pawan K. Dadheech Lothar Krienitz Kiplagat Kotut reas Ballot & Peter Casper 《FEMS microbiology ecology》2009,68(3):340-350
PCR-based denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments was used to identify the cyanobacterial phylotypes in sediments and plankton of saline–alkaline and freshwater lakes of Kenya. The detection of the aminotransferase domain located on modules mcyE and ndaF using specific molecular markers confirmed the presence of potential toxin-producing cyanobacteria. The eight nucleotide sequences obtained from DGGE bands were placed in three divergent cyanobacterial clusters. Five nucleotide sequences were close to members of the genera Anabaenopsis and Umezakia ( Nostocales ), two sequences fell in the cluster with Arthrospira sp. ( Oscillatoriales ) and one sequence was related to Chroococcidiopsis sp. ( Pleurocapsales ). The presence of the latter taxon was demonstrated de novo in the investigated lakes. All nine attained nucleotide sequences of the aminotransferase region belonged to the mcyE module. Five sequences of the aminotransferase domain were included in the cluster having the nucleotide sequence of Anabaena sp. but showed a separate lineage. Other four aminotransferases were placed in the cluster represented by nucleotide sequence of Microcystis aeruginosa . To our knowledge, this is the first report on molecular detection of cyanobacterial phylotypes in sediments of African lakes and aminotransferase domains for cyanotoxin production from sediment samples in general. 相似文献
13.
E. A. Filipenko M. L. Filipenko E. V. Deineko V. K. Shumnyi 《Cytology and Genetics》2007,41(4):199-203
DNA fragments containing T-DNA/plant DNA junctions isolated from 17 transgenic tobacco plants were amplified using inverse PCR. Analysis of the nucleotide sequences of 34 cloned DNA fragments revealed 100% homology with vector sequences outside T-DNA in 10 cases. Nine nucleotide sequences had homology with the repeats in the tobacco genome. The percentage of homology varied from 70 to 90%, with the identified repeats belonging to different types. In most clones no homology was revealed with the GENEBANK sequences. Alignment of the sequences truncated during the integration of the left and the right borders of the T-DNA insertions demonstrated significant clusterization (10 bp region) of truncation sites for the left border. Five sequences had identical truncation sites (+23 T) that showed the perferable use of this nucleotide. The AT content varied from 51 to 72% which was close to the total percentage of AT pairs in the tobacco genome. 相似文献
14.
Damaj MB Beremand PD Buenrostro-Nava MT Ivy J Kumpatla SP Jifon J Beyene G Yu Q Thomas TL Mirkov TE 《Génome》2010,53(10):840-847
The availability of a wider range of promoters for regulated expression in valuable transgenic crops would benefit functional genomics studies and current biotechnology programs aimed at improved productivity. Polymerase chain reaction (PCR)-based genome walking techniques are commonly used to isolate promoters or 5' flanking genomic regions adjacent to known cDNA sequences in genomes that are not yet completely sequenced. However, these techniques are problematic when applied directly to DNA isolated from crops with highly complex and large genomes. An adaptor ligation-mediated PCR-based BAC genome walking method is described here for the efficient isolation of promoters of multigene family members, such as the putative defense and fiber biosynthesis DIRIGENT genes that are abundant in the stem of sugarcane, a species with a highly polyploid genome. The advantage of this method is the efficient and specific amplification of the target promoter using BAC genomic DNA as template for the adaptor ligation-mediated PCR walking. 相似文献
15.
16.
Frangoulidis D Meyer H Kahlhofer C Splettstoesser WD 《FEMS immunology and medical microbiology》2012,64(1):134-136
The diagnosis of Q fever (Coxiella burnetii infection) relies primarily on the serological detection of specific antibodies. Recently, PCR-based methods have been introduced in diagnostic laboratories. Unfortunately, the fastest and most reliable 'real-time' detection method, which employs the 'online' detection of target nucleotide sequences while the amplification process is still in progress, requires expensive devices and consumables. In this study, we present a simple method that combines the simplicity of conventional PCR with new technical and methodical enhancements, resulting in a fast, specific and easy method for the molecular detection of C. burnetii. A collection of C. burnetii reference strains was tested with the modified conventional gel-based PCR approach applying a particluar PCR buffer (QIAGEN(?) Fast Cycling PCR kit) and using a closed ready-to-use gel-cassette-system (FlashGel(?)) for the visualization of specific PCR products. The modified conventional PCR method reached nearly the speed of the LightCycler(?) HybProbe real-time PCR assay (120 vs. 90 min) and showed equal sensitivity and specificity. The general cost per PCR run was 25% less than that for the LightCycler method. These improvements make this method suitable for small laboratories with limited resources and for deployable PCR diagnostics in field laboratories. 相似文献
17.
Pusch CM Kayademir T Prangenberg K Conard NJ Czarnetzki A Blin N 《Journal of applied genetics》2002,43(3):351-364
C/G-->T/A nucleotide alterations have been shown to hamper the straightforward interpretation of mitochondrial DNA sequence data derived from ancient tissues. Attempting to characterise this finding with respect to nuclear DNA, we contrasted two established protocols: (i) an enzymatic repair of damaged DNA, thereby translating and closing nicks in the DNA, and (ii) the application of N-phenacylthiazolium bromide, which cleaves glucose-derived protein crosslinks, presumably derived from Maillard reactions. We used medieval human bones that were refractory to standard PCR procedures. Due to negligible presence of short tandem repeat loci and also mitochondrial sequences, the extracted ancient DNA needed a higher copy PCR system to yield amplification products. The chosen PCR target was specific alphoid repetitive DNA with an experimentally determined minimum of 1000 copies per haploid genome. Alphoid repeat segments were generated from both contemporary DNA and DNA extracts of two human skeletons dating from 450-600 AD (omitting uracil N-glycosylase pre-treatment of the extracted samples), and were subsequently cloned and sequenced. The sequences were evaluated for the number and type of nucleotide alterations noted after the different pre-treatments, and were compared to our alphoid consensus sequence generated from modern DNA. Both methods failed to reflect the expected 32% variability among single alphoid repeats (accounting for locus-specific differences and polymerase errors) as well as to display the actual 2.88 ratio of transitions to transversions. Our data obtained from high-copy-number nuclear DNA mirror the phenomenon of sequence deviations observed in mitochondrial DNA extracted from old specimens. 相似文献
18.
The present study attempts to analyse sequences of the X- and Y-chromosome specific regions of the amelogenin (AMEL) gene
in red deer. To this end, primers specific for each form of the gene (AMELX and AMELY) were designed based on bovine genomic
sequences and the homologous regions of the genes were sequenced. The obtained sequence of AMELX gene showed high similarity
with the corresponding region in cattle (91%) and humans (77%), but this similarity was slightly lower among AMELY genes and
showed 87 and 73% of identical nucleotides, respectively. In addition, three single nucleotide polymorphisms (SNPs) were found
in the AMELX gene of the female red deer investigated. Comparative analysis of the homologous fragments of the red deer AMELX
and AMELY genes confirmed the deletion of an AMELY gene fragment in relation to AMELX. Homology of both sequences was 82%
of identical nucleotides in the coding region and 74% in 3′ non-coding sequence. The sequences studied showed considerable
similarity to homologous fragments of the human and bovine gene, but the structural differences observed lead us to design
PCR-based method for sex identification in red deer, based on the presented sequences. 相似文献
19.
David A. Randolph James W. Verbsky Liping Yang Yifu Fang Razqallah Hakem Larry E. Fields 《Transgenic research》1996,5(6):413-420
Gene targeting by double homologous recombination in murine embryonic stem (ES) cells is a powerful tool used to study the cellular consequences of specific genetic mutations. A typical targeting construct consists of a neomycin phosphotransferase (neo) gene flanked by genomic DNA fragments that are homologous to sequences in the target chromosomal locus. Homologous DNA fragments are typically cloned from a murine genomic DNA library. Here we describe an alternative approach whereby the inducible nitric oxide synthase (NOS2) gene locus is partially mapped and homologous DNA sequences obtained using a long-range PCR method. A 7 kb NOS2 amplicon is used to construct a targeting vector where theneo gene is flanked by PCR-derived homologous DNA sequences. The vector also includes a thymidine kinase (tk) negative-selectable marker gene. Following transfection into ES cells, the PCR-based targeting vector undergoes efficient homologous recombination into the NOS2 locus. Thus, PCR-based gene targeting can be a valuable alternative to the conventional cloning approach. It expedites the acquisition of homologous genomic DNA sequences and simplifies the construction of targeting plasmids by making use of defined cloning sites. This approach should result in substantial time and cost savings for appropriate homologous recombination projects. 相似文献
20.
D. E. Harry B. Temesgen D. B. Neale 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,97(3):327-336
We report a strategy for developing codominant PCR-based genetic markers by using sequenced cDNA clones from loblolly pine
(Pinus taeda L.). These clones were previously used as probes for detecting restriction fragment length polymorphisms (RFLPs) to generate
linkage maps. After assessing the complexity of banding patterns from Southern blots, we selected clones representing relatively
simple gene families, and then determined nucleotide sequences for about 200 bp at each end of the cDNA inserts. Specific
PCR primers were designed to amplify samples of genomic DNA derived from two loblolly pine mapping populations. Polymorphisms
were detected after digesting the amplified DNA fragments with a battery of restriction endonucleases, and most polymorphisms
were inherited in a Mendelian fashion. These newly identified genetic markers are codominant and relatively simple to use.
By assaying DNA from individuals used to construct RFLP maps, we show that most of these markers map to the same position
as the RFLP loci detected using their corresponding cDNAs as probes, implying that these markers have been converted from
RFLP to PCR-based methods. These PCR-based markers will be useful for genome mapping and population genetics.
Received: 10 February 1998 / Accepted: 25 February 1998 相似文献